Paramyxoviruses have a non-segmented negative strand RNA as the genome. Six genes are coded in the genome, and a short sequence (E-IG-S signal) is commonly linked to each gene. These signal sequences are highly conserved especially within a genus and within a family, and is extremely high among genes of a given virus species (Feldmann, H. E. et al., 1992, Virus Res. 24:1–19).
Sendai virus (SeV) , classified into Respirovirus in the family Paramyxoviridae, is an enveloped, non-segmented negative-strand RNA virus that is considered to be the prototype for the subfamily Paramyxovirinae. The SeV genome is 15,384 bases in size, starting with a short 3′ leader region, followed by six genes encoding the N (nucleocapsid), P (phospho), M (matrix), F (fusion), HN (hemagglutinin-neuraminidase) and L (large) proteins, and ending with a short 5′ trailer region. In addition to the P protein, the second gene expresses the accessory V and C proteins by a process known as co-transcriptional editing that inserts a G residue not comprised in the template (Park, K. H. and M. Krystal, 1992, J. Virol. 66:7033–7039; Paterson, R. G., and R. A. Lamb, 1990, J. Virol. 64:4137–4145; Thomas, S. M. et al., 1988, Cell, 54:891–902; Vidal, S. et al., 1990, J. Virol. 64:239–246) and by alternative translational initiations, respectively (Gupta, K. C., and E. Ono, 1997, Biochem. J. 321:811–818; Kuronati, A. et al. , 1998, Genes Cells 3:111–124). The genome is tightly associated with the N protein, forming a helical ribonucleoprotein (RNP) complex. This RNP, but not the naked RNA, is the template for both transcription and replication (Lamb, R. A., and D. Kolakofsky, 1996, Paramyxoviridae: The viruses and their replication. pp. 1177–1204. In Fields Virology, 3rd edn. Fields, B. N., D. M. Knipe, and P. M. Howley et al. (ed.), Raven Press, New York, N.Y.) There is only a single promoter at the 3′ end for viral RNA polymerase comprising the P and L proteins (Hamaguchi, M. et al., 1983, Virology 128:105–117). By recognizing the short, conserved transcription end (E) sequence and transcription start (S) sequence at each gene boundary, the polymerase produces leader RNA and each of the mRNAs (Glazier, K. et al., 1997, J. Virol. 21:863–871). There is a trinucleotide intergenic (IG) sequence between the E sequence and S sequence, which is not transcribed (Gupta, K. C., and D. W. Kingsbury, 1984, Nucleic Acids Res. 12:3829–3841; Luk, D. et al., 1987, Virology 160:88–94). Since the efficiency of reinitiating transcription at each gene boundary is high but not perfect, the transcripts from the downstream genes are less abundant than those from the upstream genes. Therefore, each mRNA is not synthesized in equimolar quantities in infected cells, but there is a polar attenuation of transcription toward the 5′ end (Glazier, K. et al., 1997, J. Virol. 21:863–871; Homann, H. E. et al., 1990, Virology 177:131–140; Lamb, R. A., and D. Kolakofsky, 1996, Paramyxoviridae: The viruses and their replication. pp. 1177–1204. In Fields Virology, 3rd edn. Fields, B. N., D. M. Knipe, and P. M. Howley et al. (ed.), Raven Press, New York, N.Y.).
After the translation of the mRNAs and accumulation of translation products, genome replication takes place. Here, the same viral RNA polymerase conducts replication using the same RNP template, but now somehow ignores the respective E sequence and S sequence of each mRNA and generates a full length antigenomic positive sense (+)RNP (Lamb, R. A., and D. Kolakofsky, 1996, Paramyxoviridae: The viruses and their replication. pp. 1177–1204. In Fields Virology, 3rd edn. Fields, B. N., D. M. Knipe, and P. M. Howley et al. (ed.), Raven Press, New York, N.Y.). The polymerase enters the promoter at the 3′ end of (+)RNP to generate genomic (−) RNP, which serves as the template for the next round of transcription and replication.
The B sequence (3′-AUUCUUUUUU-5′ (SEQ ID NO: 26) in the genomic negative sense) is completely conserved among the six genes in the SeV genome. The five U residues in the latter half are thought to allow the polymerase slippage-generating poly(A). In contrast, the S sequences are slightly varied and are generalized as 3′-UCCCWVUUWC-5′ (SEQ ID NO: 27) (Gupta, K. C., and D. W. Kingsbury, 1984, Nucleic Acids Res. 12:3829–3841). Specifically, the S sequence is UCCCACUIJUC (SEQ ID NO: 28) for P, M and HN genes, UCCCAgUUUC (SEQ ID NO: 29) for N gene, UCCCuaUUUC (SEQ ID NO: 30) for F gene, and UCCCACUUaC (SEQ ID NO: 31) for L gene. Identical differences are seen in all SeV strains sequenced to date, regardless of differences in isolation procedure, passage history, and virulence for the natural host such as mice, suggesting that the variations are locus-specific. It is possible that these differences arise as a result of nucleotide accumulation in sites that are unaffected by variations in the S sequence. Another possibility is that these differences arise due to nucleotide substitutions at important sites of the signal and the selection of viruses that have acquired the ability to regulate the expression of each gene during viral evolution.
Up to now, several studies with model template systems of various nonsegmented negative strand RNA viruses have indicated that the S sequences are indeed critical for transcriptional initiation, but sequence variations are tolerated to some extent (Barr, J. N. et al., 1997, J. Virol. 71:1794–1801; Barr, J. N. et al., 1997, J. Virol. 71:8718–8725; Hwang, L. N. et al., 1998, J. Virol. 72:1805–13; Kuo, L. et al., 1996, J. Virol. 70:6143–6150; Rassa, J. C., and G. D. Parks, 1998, Virology, 247: 274–286; Stillman E. A., and M. A. Whitt, 1998, J. Virol. 72: 5565–5572). Certain nucleotide substitutions in these S sequences were shown to decrease transcription initiation efficiency, suggesting that gene expression is also modulated by naturally occurring variations in the viral life cycle. (Kuo, L. et al., 1996, J. Virol. 70:6892–6901; Kuo, L. et al., 1997, J. Virol. 71:4944–4953; Stillman E. A., and M. A. Whitt, 1997, J. Virol. 71:2127–2137). However in the model template systems, all events required early in the natural life cycle like primary transcription is by-passed by the successive and constant supply of trans-acting proteins (Nagai, Y. Paramyxovirus replication and pathogenesis. Reverse genetics transforms understanding. Rev. Medical. Virol. 9(2): 83–99 (1999)). The transcription and replication of minigenomes are uncoupled in these systems. T7 polymerase-expressing vaccinia virus often used to produce tans-acting proteins masks the subtle effects of mutations by, for example, posttranscriptional modifications by capping enzymes encoded by vaccinia viruses. In addition, transfection efficiencies might not be equal throughout the whole experiment (Bukreyev, A. et al., 1996, J. Virol. 70:6634–6641; He, B. et al., 1997, Virology 237:249–260). Namely, effects of nucleotide substitutions in the S sequence on transcription initiation cannot be accurately examined in model template systems. Thus, to comprehensively evaluate the roles of S sequence and E sequence, it was necessary to introduce mutations into the full-length viral genome.